Design and Characterization of Light-Inducible Tyrosine Phosphatases to Tune Synthetic Receptor Tyrosine Kinase Signaling

Abstract

Transmembrane receptor proteins are essential for translating extracellular signals into programmed cellular behaviors. Synthetic Receptor Tyrosine Kinases (RTKs) bearing non-native extra- and intracellular domains have emerged as powerful tools for activating novel cell behaviors in response to specified inputs. Endogenous RTK signaling achieves a level of precision in both space and time that synthetic RTKs have yet to display, owing largely to their tight regulation by Protein Tyrosine Phosphatases (PTPs). To achieve precise, tunable signaling with synthetic RTKs, we engineered synthetic PTPs that function as programmable regulators of Epidermal Growth Factor Receptor (EGFR) phosphorylation. We first generated a panel of synPTPs that bind to an auxiliary phosphotyrosine motif on the tail of EGFR in response to its activation with a ligand. We found some of these synPTPs dephosphorylate the auxiliary motif near-completely, but the EGFR-driven binding of these synPTPs perpetually attenuated EGFR phosphorylation. We then generated a light-inducible optoPTP platform for recruiting phosphatase catalytic domains to EGFR independently of EGFR activation. optoPTP1B displays light-inducible dephosphorylation of EGFR on the scale of minutes. optoPTP1B did not, however, completely dephosphorylate cellular EGFR, and we observed no significant change to EGFR-driven ERK activation in response to light-induced PTP recruitment. Additionally, we did not observe any off-target disruption of PDGFR signaling by optoPTP1B. Our synPTP and optoPTP systems demonstrate that engineering the recruitment of phosphatase catalytic domains to the tail of an RTK is a promising strategy for precisely controlling the dynamics of RTK signaling in both space and time.